In plaques, found in Alzheimer's disease (AD), only a small proportion of Aβ peptides begin with an N-terminal aspartate (AβN1D). The majority starts at position 3 with pyroglutamate (AβN3(pGlu))(Kuo, Y. M., Emmerling, M. R., Woods, A. S., Cotter, R. J. & Roher, A. E. Isolation, chemical characterization, and quantitation of Abeta 3-pyroglutamyl peptide from neuritic plaques and vascular amyloid deposits. Biochem Biophys Res Commun 237, 188-191. (1997); Saido, T. C., et al. Dominant and differential deposition of distinct beta-amyloid peptide species, AbetaN3(pE), in senile plaques. Neuron 14, 457-466 (1995)), and ends at position 42. Aβ starting with N-terminal glutamine (AβN3Q) is a better substrate for cyclization by glutaminyl cyclase (QC) than Aβ starting with N-terminal glutamate (AβN3E), (Schilling, S., Hoffmann, T., Manhart, S., Hoffmann, M. & Demuth, H. U. Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions. FEBS Lett 563, 191-196 (2004); Cynis, H., et al. Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells. Biochim Biophys Acta 1764, 1618-1625 (2006)).
Aβ(N3pGlu) has a higher aggregation propensity (He, W. & Barrow, C. J. The Abeta 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater beta-sheet forming and aggregation propensities in vitro than full-length Abeta Biochemistry, 38, 10871-10877 (1999); Schilling, S., et al., On the seeding and oligomerization of pGlu-amyloid peptides (in vitro), Biochemistry, 45, 12393-12399 (2006)) and stability (Kuo, Y. M., Webster, S., Emmerling, M. R., De Lima, N. & Roher, A. E. Irreversible dimerization/tetramerization and post-translational modifications inhibit proteolytic degradation of Abeta peptides of Alzheimer's disease. Biochim Biophys Acta 1406, 291-298 (1998)), and shows an increased toxicity compared to full-length Aβ (Russo, C., et al. Pyroglutamate-modified amyloid-peptides—A N3(pE)—strongly affect cultured neuron and astrocyte survival, Journal of Neurochemistry 82, 1480-1489 (2002)). However, other studies reported that the toxicity of Aβ(N3pGlu-40) and Aβ(N3pGlu-42) is similar to that of Aβ (N1D-40) and AβN1D-42)(Tekirian, T. L., Yang, A. Y., Glabe, C. & Geddes, J. W. Toxicity of pyroglutaminated amyloid beta-peptides 3(pE)-40 and -42 is similar to that of Abeta1-40 and -42, J Neurochem 73, 1584-1589 (1999)), and that Aβ(N3pGlu) is not the major variant in Aβ brain (Lernere, C. A., et al. Sequence of deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation, Neurobiol Dis 3, 16-32 (1996)). Schilling et al. have demonstrated that pyroglutamate-modified peptides display an up to 250-fold acceleration in the initial formation of Aβ aggregates (Schilling, S., et al., On the seeding and oligomerization of pGlu-amyloid peptides (in vitro), Biochemistry, 45, 12393-12399 (2006)), and presented in vitro evidence that the cyclization of glutamate at position 3 of Aβ is driven enzymatically by glutaminyl cyclase (QC) (Schilling, S., Hoffmann, T., Manhart, S., Hoffmann, M. & Demuth, H. U. Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions, FEBS Lett 563, 191-196 (2004); Cynis, H., et al. Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells, Biochim Biophys Acta, 1764, 1618-1625 (2006)). QC inhibition leads to significantly reduced Aβ(N3pGlu) formation, showing the importance of QC-activity during cellular maturation of pyroglutamate-containing peptides (Cynis, H., et al. Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells, Biochim Biophys Acta 1764, 1618-1625 (2006)). APP transgenic mouse models have been reported to show no (Kuo, Y. M., et al. Comparative analysis of amyloid-beta chemical structure and amyloid plaque morphology of transgenic mouse and Alzheimer's disease brains. J Biol Chem 276, 12991-12998 (2001)) or low Aβ(N3pGlu) levels (Guntert, A., Dobeli, H. & Bohrmann, B. High sensitivity analysis of amyloid-beta peptide composition in amyloid deposits from human and PS2APP mouse brain. Neuroscience. 143, 461-475 (2006)), in contrast to the APP/PS1KI mouse, which harbours considerable amounts of Aβ(N3pGlu) detected by 2D-gel electrophoresis of whole brain lysates (Casas, C., et al. Massive CA1/2 Neuronal Loss with Intraneuronal and N-Terminal Truncated A{beta}42 Accumulation in a Novel Alzheimer Transgenic Model. Am J Pathol 165, 1289-1300 (2004)) and by immunohistochemistry within neurons and plaques (Wirths, O., Weis, J., Kayed, R., Saido, T. C. & Bayer, T. A. Age-dependent axonal degeneration in an Alzheimer mouse model, Neurobiol Aging 8, online version (2006)). The APP/PS1KI mice develop age-dependent axonal degeneration in brain and spinal cord (Wirths, O., Weis, J., Kayed, R., Saido, T. C. & Bayer, T. A. Age-dependent axonal degeneration in an Alzheimer mouse model, Neurobiol Aging 8, online version (2006)), a 50% neuron loss in CA1 at 10 months of age (Casas, C., et al. Massive CA1/2 Neuronal Loss with Intraneuronal and N-Terminal Truncated A {beta}42 Accumulation in a Novel Alzheimer Transgenic Model. Am J Pathol 165, 1289-1300 (2004)), and deficits in working memory and motor performance at 6 months of age (Wirths, O., Breyhan, H., Schafer, S., Roth, C. & Bayer, T. A. Deficits in working memory and motor performance in the APP/PS Iki mouse model for Alzheimer's disease, Neurobiol Aging 8, 8 (2007)). Between 2 and 6 months of age, the rate of Aβ(N3pGlu) aggregation was higher than the rate of unmodified Aβ(N1D). Although suggestive, it is difficult to correlate between Aβ(N3pGlu) deposition and the observed CA1 neuron loss in this model, due to the larger heterogeneity of N-truncated Aβ peptides (Casas, C., et al. Massive CA1/2 Neuronal Loss with Intraneuronal and N-Terminal Truncated A {beta}42 Accumulation in a Novel Alzheimer Transgenic Model. Am J Pathol 165, 1289-1300. (2004)).
Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid (pGlu*) under concomitant liberation of ammonia. A QC was first isolated by Messer from the Latex of the tropical plant Carica papaya in 1963 (Messer, M. 1963 Nature 4874, 1299). 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). For the mammalian QCs, the conversion of N-terminal Gln into pGlu by QC has been shown for the precursors of TRH and GnRH (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). In addition, initial localization experiments of QC revealed a co-localization with its putative products of catalysis in bovine pituitary, further improving the suggested function in peptide hormone synthesis (Bockers, T. M. et al. 1995 J Neuroendocrinol 7, 445-453). In contrast, the physiological function of the plant QC is less clear. In case of the enzyme from C. papaya, a role in the plant defence against pathogenic microorganisms was suggested (El Moussaoui, A. et al. 2001 Cell Mol Life Sci 58, 556-570). Putative QCs from other plants were identified by sequence comparisons recently (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36). The physiological function of these enzymes, however, is still ambiguous.
The QCs known from plants and animals show a strict specificity for L-Glutamine in the N-terminal position of the substrates and their kinetic behavior was found to obey the Michaelis-Menten equation (Pohl, T. et al. 1991 Proc Natl Acad Sci USA 88, 10059-10063; Consalvo, A. P. et al. 1988 Anal Biochem 175, 131-138; Gololobov, M. Y. et al. 1996 Biol Chem Hoppe Seyler 377, 395-398). A comparison of the primary structures of the QCs from C. papaya and that of the highly conserved QC from mammals, however, did not reveal any sequence homology (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36). Whereas the plant QCs appear to belong to a new enzyme family (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36), the mammalian QCs were found to have a pronounced sequence homology to bacterial aminopeptidases (Bateman, R. C. et al. 2001 Biochemistry 40, 11246-11250), leading to the conclusion that the QCs from plants and animals have different evolutionary origins.
EP 02 011 349.4 discloses polynucleotides encoding insect glutaminyl cyclase, as well as polypeptides encoded thereby. This application further provides host cells comprising expression vectors comprising polynucleotides of the invention. Isolated polypeptides and host cells comprising insect QC are useful in methods of screening for agents that reduce glutaminyl cyclase activity. Such agents are described as useful as pesticides.
The subject matter of the present invention is particularly useful in the field of Aβ-related diseases, one example of those being Alzheimer's Disease. Alzheimer's disease (AD) is characterized by abnormal accumulation of extracellular amyloidotic plaques closely associated with dystrophic neurones, reactive astrocytes and microglia (Terry, R. D. and Katzman, R. 1983 Ann Neurol 14, 497-506; Glenner, G. G. and Wong, C. W. 1984 Biochem Biophys Res Comm 120, 885-890; Intagaki, S. et al. 1989 J Neuroimmunol 24, 173-182; Funato, H. et al. 1998 Am J Pathol 152, 983-992; Selkoe, D. J. 2001 Physiol Rev 81, 741-766). Amyloid-beta (abbreviated as Aβ) peptides are the primary components of senile plaques and are considered to be directly involved in the pathogenesis and progression of AD, a hypothesis supported by genetic studies (Glenner, G. G. and Wong, C. W. 1984 Biochem Biophys Res Comm 120, 885-890; Borchelt, D. R. et al. 1996 Neuron 17, 1005-1013; Lernere, C. A. et al. 1996 Nat Med 2, 1146-1150; Mann, D. M. and Iwatsubo, T. 1996 Neurodegeneration 5, 115-120; Citron, M. et al. 1997 Nat Med 3, 67-72; Selkoe, D. J. 2001 Physiol Rev 81, 741-766). Aβ is generated by proteolytic processing of the β-amyloid precursor protein (APP) (Kang, J. et al. 1987 Nature 325, 733-736; Selkoe, D. J. 1998 Trends Cell Biol 8, 447-453), which is sequentially cleaved by β-secretase at the N-terminus and by γ-secretase at the C-terminus of Aβ (Haass, C. and Selkoe, D. J. 1993 Cell 75, 1039-1042; Simons, M. et al. 1996 J Neurosci 16 899-908). In addition to the dominant Aβ peptides starting with L-Asp at the N-terminus (Aβ1-42/40), a great heterogeneity of N-terminally truncated forms occurs in senile plaques. Such shortened peptides are reported to be more neurotoxic in vitro and to aggregate more rapidly than the full-length isoforms (Pike, C. J. et al. 1995 J Biol Chem 270, 23895-23898). N-truncated peptides are known to be overproduced in early onset familial AD (FAD) subjects (Saido, T. C. et al. 1995 Neuron 14, 457-466; Russo, C, et al. 2000 Nature 405, 531-532), to appear early and to increase with age in Down's syndrome (DS) brains (Russo, C. et al. 1997 FEBS Lett 409, 411-416, Russo, C. et al. 2001 Neurobiol Dis 8, 173-180; Tekirian, T. L. et al. 1998 J Neuropathol Exp Neurol 57, 76-94). Finally, their amount reflects the progressive severity of the disease (Russo, C. et al. 1997 FEBS Lett 409, 411-416; Guntert, A. et al. 2006 Neuroscience 143, 461-475). Additional post-translational processes may further modify the N-terminus by isomerization or racemization of the aspartate at position 1 and 7 and by cyclization of glutamate at residues 3 and 11. Pyroglutamate-containing isoforms at position 3 [AβN3(pGlu)-40/42] represent the prominent forms—approximately 50% of the total Aβ amount—of the N-truncated species in senile plaques (Mori, H. et al. 1992 J Biol Chem 267, 17082-17086, Saido, T. C. et al. 1995 Neuron 14, 457-466; Russo, C. et al. 1997 FEBS Lett 409, 411-416; Tekirian, T. L. et al. 1998 J Neuropathol Exp Neurol 57, 76-94; Geddes, J. W. et al. 1999 Neurobiol Aging 20, 75-79; Harigaya, Y. et al. 2000 Biochem Biophys Res Commun 276, 422-427) and they are also present in pre-amyloid lesions (Lalowski, M. et al. 1996 J Biol Chem 271, 33623-33631). The accumulation of AβN3(pE) peptides is likely due to the structural modification that enhances aggregation and confers resistance to most amino-peptidases (Saido, T. C. et al. 1995 Neuron 14, 457-466; Tekirian, T. L. et al. 1999 J Neurochem 73, 1584-1589). This evidence provides clues for a pivotal role of AβN3(pE) peptides in Aβ pathogenesis. However, little is known about their neurotoxicity and aggregation properties (He, W. and Barrow, C. J. 1999 Biochemistry 38, 10871-10877; Tekirian, T. L. et al. 1999 J Neurochem 73, 1584-1589). Moreover, the action of these isoforms on glial cells and the glial response to these peptides are completely unknown, although activated glia cells are strictly associated to senile plaques and might actively contribute to the accumulation of amyloid deposits. In recent studies the toxicity, aggregation properties and catabolism of Aβ1-42, Aβ1-40, [pGlu3]Aβ3-42, [pGlu3]Aβ3-40, [pGlu11]Aβ11-42 and [pGlu11]Aβ11-40 peptides were investigated in neuronal and glial cell cultures, and it was shown that pyroglutamate modification exacerbates the toxic properties of Aβ-peptides and also inhibits their degradation by cultured astrocytes. Shirotani et al. investigated the generation of [pGlu3]Aβ peptides in primary cortical neurons infected by Sindbis virus in vitro. They constructed amyloid precursor protein complementary DNAs, which encoded a potential precursor of [pGlu3]Aβ by amino acid substitution and deletion. For one artificial precursor starting with an N-terminal glutamine residue instead of glutamate in the natural precursor, a spontaneous conversion or an enzymatic conversion by glutaminyl cyclase to pyroglutamate was suggested. The cyclization mechanism of N-terminal glutamate at position 3 in the natural precursor of [pGlu3]Aβ was neither determined in vitro, in situ nor in vivo (Shirotani, K. et al. 2002 NeuroSci Lett 327, 25-28).